Lecture 6: Enzymes - Catalytic Strategies Flashcards
Factors that influence enzyme activity
- temperature
- pH
- enzyme and substrate concentration
- reaction complexity (bisubstrate, ping pong, etc)
- inhibitors (and activators)
Temperature and enzyme activity
- higher temperature, increases activity (only up to optimum temp for enzymes)
- above optimum the reaction rate decreases and enzyme loses shape or denatures (usually irreversible)
pH and enzyme activity
- reaction rate increases as pH nears optimum level
- above or below, enzyme function can be disrupted and reaction rate decreases
Enzyme activity and substrate concentration
- rxn rate increases with increased amounts of enzyme or substrate
- only up to the point at which all of the enzyme molecules are bound to the substrate
- at this point, additional enzyme (or substrate) no longe rincreases the rxn rate
Bisubstrate rxns
- most reactions in biological systems start with two substrates and yield two products
- transfer of a functional group from one substrate to another
Two classes:
- sequential reactions (random or ordered)
- double displacement reactions (ping pong)
Random sequential mechanisms
Example
- enzyme and substrates can combine in any order
Creatine –> phospohcreatine
- mitochondria normally makes ATP until equilibrium
- creatine makes ATP in cytosol low (binds it)
- mitochondria then makes more ATP
- in physical activity the reserves from ohosphocreatine are used up first
Ordered sequential mechanisms
- must be combined in a certain order
- enzyme needs to bind coenzyme before substrate binds
puruvate + NADH –> lactate + NAD+
Kinetics of a sequential mechanism
Double displacement or ping pong mechanisms
- transfers groups between molecules
- 1st reaction fast, 2nd slow
- often in formation of amino acids –> trasnfers amino grops between several molecules
Kinetics of a double displacement mechanism
slope stays the same
vmax increases as substrate concentration increases
Inhibited reactions
What do inhibitors do?
- an enzyme inhibitor is a compound that binds to an enzyme and interferes with its activity by decreasing its reaction rates
- an inhibitor can act by preventing the formation of an ES complex or by blocking a chemical reaction that leads to the formation of a product
- inhibited reactions can be reversible or irreversible
Types of reversible inhibition
- Competitive: inhibitor can bind only to free enzyme molecules that have not bound any substrate
–> association raises Km, while Vmax remains unchanged
- Uncompetitive inhibition: inhibitor binds only to ES and not to free enzyme
–> association lowers Km and Vmaz, while the ration of Vmax /Km remains unchanged
- Noncompeitive: inhibitor can bind to E or ES forming inactive EI or ESI complexes, respectively
–> association lowers Vmax while Km remins unchanged (or nearly unchanged)
Irreversible inhibition
- inhibitor forms a stable covalent bond with an enzyme molecule, thus removing active molecules from the enzyme population
- irreversible inhibition typically occurs by alkylation or acylation of the side chain (R-group) of an active site amino acid residue
Group Specific reagents as irreversible inhibitors
Example?
- inhibit enzyme by reacting with specific side chains of amino acids that can be crucial for enzyme activity
- enzyme acetylcholine esterase is needed to remove acetyl choline
- acetyl choline triggers release of Ca2+ from stores in cells that then lead to muscle contraction
- if acetylcholine not removed, muscles are stuck contracting
- acetylcholine has a serine that gets inactivated by DIPF
Affinity labels as irreversible inhibitors
reactive substrate analogs are molecules that are structurally similar to the substrate for an enzyme and covalently bind to its ative site residue
Transition state analogs as irreversible inhibitors
Convert from L proline to D-proline
Transition state analogs and human health
- enzymes are often targets for test drugs and other beneficial agents
- transition state analogs often make ideal (rev and irrev) enzyme inhibitors
ex:
- statins lower cholesterol
- juvenile hormone esterase (JHE) is a pesticide target
Statins
- powerful cholesterol lowering drugs
- transition state analog inhibitors of HMG-CoA reductase, a key enzyme in the biosynthetic pathway for cholesterol
Need for Juvenile hormone esterase (JHE) Inhibitors
- insects have significant effects on human health
- malaria, west nile, viral encephalitis carried by mosquitos
- lyme disease and rocky mountain fever carried by ticls
Controlling insect population strategy
- alter the actions of juvenile hormone, a terpene-based substance that regulates insect life cycle processes
- levels of juvenile hormone are controlled by JHE and inhibition of JHE is toxic to insects
- OTFP is a potent transition state analog inhibitor of JHE
Suicide inhibitors
- mechanism based inhibitors bind to an enzyme as a substrate
- mechanism of catalysis then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification
How does penicillin work?
covalently bonds glycopeptide transpeptidase
Competitive inhibition graph
- same vmax
- different Km
uncompetitive inhibition graph
- km and vmax both change
- ration doesnt change
Noncompetitive inhibition graph
- earlier saturation
- lower than qithout inhibitor
How do enzymes act as catalysts
- enzymes lower the activation energy by stabilizing the transition state with respect to the uncatalyzed reaction
- enzymes are catalysts due to their specificity of substrate binding combined with their optimal arrengement of catalytic groups
General strategies of enzyme catalysis
- approximation and orientation effects
- acid-base catalysis (protonation/deprotonation)
- covalent catalysis
- metal ion catalysis (often iron)
- preferential binding of the transition state complex
Catalysis through proximation and orientation effects
- anzymes utilize catalytic mechanisms that resemble those of organic model reactions but are far more catalytically effecient than these models
- efficiency arises from specific conditions at the catalytic or active sites that promote the corresponding chemical reactions
- proximity and orientation effects contribute to such conditions as enzymes associate substrates in a manner that both aligns and immobilizes substrates so as to optimize reactivities
* might involve other strategies like acid/base but this allows them to happen
Acid base catalysis
Two types:
- specific acid base catalysis - reaction is accelerated by H+ or OH- diffusing in from the solution (already around)
- general acid base catalysis - H+ or OH- is created in the transition state by another molecule or group, which is termed the general acid or general base, respectively (proton is transferred in the TS) (made during the process)
General acid catalysis
- general acid: process in which partial proton transfer from a proton-donating acid lwers the free energy of a reactions transition state
* many enzymatic reactions are subject to both processes
general base catalysis
- a reaction can also be stimulated by general base catalysis if its rate is increased by partial proton abstraction by a proton-accepting base
Keto enol tautomerism
- example of both general acid and base catalysis
amino acids involved in general acid-base catalysis
GLU, ASP (COOH groups)
LYS, ARG (NH groups)
CYS (s- H)
Hist (H on N of ring)
Ser (OH group)
Tyr (OH on ring)
covalent catalysis
- many enzymatic reactions derive much of their rate acceleration from the transient formation of a covelant bond between enzyme and substrate
BY + X –> BZ+ Y
with enzyme:
BY + E-X –> E-X:B + Y + Z –> E-X + BZ (E = enzyme)
Three stages of catalysis
- nucleophilic reaction: between catalyst and the substrate to form covalent bond
- withdrawal of electrons: fromt he reaction center by the now electrophilic catalyst
- elimination of the catalyst: a reaction that is essentially the reverse of stage 1
Nucleophilic and electrophilic centers
- side chains of amino acids in enzymes (active sites) offer a variety of nucleophilic centers for catalysis (including amines, carboxylates, aryl and alkyl hydroxyls, imidazoles and thiol groups)
- these groups readily attack electrophilic centers of substrates (phosphoryl groups, acyl groups, glycosyl groups) forming the covalently bonded enzyme substrate intermediates
- intermediates are then attacked by a water molecule or a second substrate, giving the desired product
metal ion catalysis
- nearly 1/3 of all known enzymes depend on the presence of metal ions for (full) catalytic activity
- there are 2 classes of such enzymes that are distinguished by the strengths of their ion protein interactions
1. metalloenzymes - contain tightly bound metal ions, most commonly transition metal ions such as Fe2/3+, Cu+/2+, Zn2+, Mn2+, Co3+
2. metal-activated enzymes- loosely bind metal ions from solution, usually the alkalai and alkaline earth metal ions Na+, K+, Mg2+, Ca2+
How do metal ions participate in catalytic processes
- serve as a bridge between enzyme and substrate to increase the binding energy and to hold the substrate in a conformation appropriate for catalysis
- electrostatically stabilize or shield negative charge in the enzyme’s active site or on a reaction intermediate
- facilitate the formation of nucleophiles such as hydroxide ion (OH-) from water at (near) neutral pH by direct coordination
- mediating redox reactions through reversible changes in the metal ions oxidation state
Thermolysin
- endoprotease with a Zn2+ ion in its active site, functioning as electrophilic catalyst
- stabilizes the buildup of negative charge on the peptide carbonyl oxygen, as a glutamate residue deprotonates water, promoting hydroxide attack on the carbonyl atom
- Zn2+ allows for general acid catalysis
Preferential binding of the TS complex
- all enzymes bind transition states with higher affinity than the corresponding substrates pr products
- either enzymes mechanically strain their substrates toward the transition state geometrythrough binding sites into which undistorted substrates do not properly fit
- OR substrates induce conformational changes in corresponding enzymes upon binding and these conformational changes result in favorable formation of transition states
* enzyme and substrate are not a perfect fit… enzyme forces substrate into TS to be a good fit
–> induced fit model
Lock and key vs induced fit
induced fit preferentially binds the TS state and thus proportionally increases the reaction rate
Importance of carbonic anhydrase
- major end product of aerobic metabolism
- in mammals this CO2 is released into the blood and transported to the lungs for exhalation
–> while in red blood cells (erythrocytes), CO2 reacts with H2O to form carbonic acid (H2CO3) which is then converted to bicarbonate ion HCO3-
- HCO3- diffuses towards lungs where it is in lowest concentration
- lungs are then low in CO2 so HCO3- is converted to CO2 and exhaled out
Carbonic anhydrase mechanism
what is carbonic anhydrase?
What are metalloenzymes?
- metalloenzyme containing a zinc ion essential for its catalytic mechanism
–> metal ions have several properties that increase chemical reactivity, such as their positive charges, their ability to form strong yet kinetically labile bonds, and in some cases, their capacity to be stable in more than one oxidation state
–> about 1/3 of all enzymes either contain bound metal ions or require their addition for full activity
- one zinc ion per enzyme is bound to the imidazole rings of three histidine residues as well as to a water molecule
Carbonic anhydrase:
How does Zn2+ facilitate water deprotonation
- Zn2+ facilitates the proton rleease from water by lowering the pKa of the ater molecule (water deprotonation)
- CO2 substrate binds to the enzymes active site and is positioned to react with the OH- ion (carbon dioxide binding)
Carbonic anhydrase: nucleophilic attack
- OH- attacks the CO2 converting it into HCO3- (nucleophilic attack)
- the catalytic site is regenerated with the release of HCO3- and the binding of another molecule of water (displacement of carbonate ion by water)
Zinc bound hydroxide mechanism for carbon dioxide hydration
pH and carbonic anhydrase
- feedback mechanism in which enzyme can sense pH
- makes the weak acid, and if makes enough, it loses activity
- when there is less bicarbonate more water deprotonation occurs and more bicarbonate produced
What is lysozyme
- a natural antibacterial agent that cleaves the B(1–>4) glycosidic linkages from N-acetylmuramic acid (NAM) to N-acetylglucosamine (NAG) in the alternating NAM-NAG polysaccharide component of bacterial cell wall peptidoglycan
- specifically cleaves between the 4th and 5th sugar of a hexosaccharide
Lysozyme binding and hydrolysis of peptidoglycan
- uses water to force open the bond between the sugars
Lysozyme mechanism
Lysozyme mechanism steps
1a. binding to a NAM-NAG hexasaccharide (A-F) unit
1b. orietning the D and E rings in the catalytic site
2a. Proton transfer of GLU35 to the O1 atom linking the D and E rings
2b. Cleavage of the C1-O1 bond (general acid catalysis) and release of the E ring product
3a. Nucleophilic attack of the D ring C1 by Asp52
3b. Formation of a covalent glycosyl-enzyme intermediate (covalent catalysis)
4. Glu35 now acts as a general base catalyst to form a hydroxide (Oh-) ion from water
5. OH- displaces Asp52 to generate the d ring
* main idea is that the polysaccharide is initially cleaved, one piece is let go, then the other end remains and is further modified and let go later - so that the two pieces cannot reform
lysozyme activity vs pH
most active between 4 and 6 pH
Serine proteases
- protein turnover is an important process in living systems:
–> proteins ingested in the diet must be broken down into small peptides and amino acids for absorption in the gut (processing dietary proteins)
–> proteins that have served their purpose must be degraded so that their constituents (amino acids) can be recycled for the synthesis of new proteins (recycling amino acids)
–> proteolytic reaction are involved in regulating the activity of enzymes and other proteins (regulation)
* we cant make some aas, so need to get them from other proteins
Cleaving proteins by hydrolysis reaction
- proteases in general cleave proteins by a hydrolysis reaction (add water molecule to a peptide bond)
- although the hydrolysis of peptide bonds is thermodynamically favored, it is extremely slow in the absence fo catalyst
chymotrypsin
- serine protease
- cleaves peptide bonds selectively on the carboxyl-terminal side of large hydrophobic amino acids such as trp, Tyr, Phe and Met
Chymotripsin and serine residue
- treated with DIPF (dispropylphosphoflouridate - acts and irreversible inhibitor) it loses all activity although only a single residue (Ser195) was modified
–> suggests that this unusually reactive Ser plays a central role in the catalytic mechanism
Kinetic characteristics of chymotrypsin
- many proteases (including chymotrypsin) can act as a protease as wel as an esterase
–> the chemical mechanisms of ester and amide hydrolysis are almost identical
- a chromogenic substrate is used to monitor esterase activity (yielding yellow product)
kinetic characteristics of phases of ester hydrolysis
- 1st substrate is nitrophenylacetate
- second is H2O
- nitrophenolate is produced very fast at first
- acetate is produced slowly
covalent catalysis and chymotrypsin
catalytic triade and oxyanion hole
- Asp, hist and Ser
- asp draws from hist, and hist draws from serine to make serine an alkoxide ion
catalytic triade and oxyanion hole - TS stabilization
- the transition state fits perfectly into the oxyanion hole
Serine proteases and convergent evolution
there are a number of serine proteases
- demonstrate convergent evolution
- only thing in common is catalytic triade
- conserved in function but not sequence
serine proteases: from zymogen to active forms
- not active initially
- mus tbe activated
cysteine proteases
- exhibit a catalytic mechanism similar to serine proteases
–> instead of a serine residue, a cysteine residue (activated by a histidine) playys the role of the nucleophile that attacks the peptide bond
–> because the sulfu atom in the cystein is inherently a better nucleophile than is the oxygen atom in serine, cysteine proteases appear to require only this histidine residue in addition to cysteine and not the fully cataltic triad
- examples are papain (papaya fruit) and animal lysozymes (cathespins)
List of nonserine proteases
- metalloproteases
- aspartic proteases
metalloproteases
- have active sites that contain a bound metal ion, almost always zinc, which activates a water molecule to serve as a nucleophile to attack the peptide carbonyl group
- examples of zinc proteases are the bacterial enzyme thermolysin, the digestive enzyme carboxypeptidase A, and various matrix metalloproteases (MMPs) that are involved in degradation of the extracellular matrix during tissue remodeling
Aspartic proteases
- the central feature of their active sites is a pair of aspartic acid residues that act together to allow a water molcule to attack the peptide bond
–> one aspartic acide residue (in its deprotonated form) activates the attacking water molecules by poising it for deprotonation
–> the other aspartic acid (in itsprotonated form) polarizes the peptide carbonyl group so that it is more susceptible for attack
examples of aspartic proteases
- pepsin (stomach - dietary proteins)
- chymosin (stomach- dietary proteins)
- cathespin D (spleen, liver, and many other animal tissues)
- renin (kinday)
- HIV- protease (AIDS virus)
catalytic mechanism of HIV protease
cleaves peptides of HIV
- water attacks carbonyl carbon, generating a tetrahedral intermediate stabilized by H bonding
- tetrahedral intermediate collapses; the amino acid leaving group is protonated as it is expelled
Designing enzyme inhibitors
- aspartic proteases are the target
